An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins

Abstract

In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin-agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

“…Furthermore, early information about rFNIII9/10 solubility is accurately identifiable by following the coloration of the soluble fraction and the harvested cell pellet without resorting to the time-consuming steps of gel analysis (Figure A1). As we have demonstrated previously [40], a direct correlation was found between the red coloration intensity of fractions and produced protein with an optical density (OD) profile at 400 nm measurement of each sample. This system allows a rapid and efficient method to monitor protein expression at different points of time during production (Figure 1).…”

“…During production, the soluble expression of rFHIII9/10 and biomass were evaluated in real time due to the CMAT fusion partner and was analyzed by SDS-PAGE. As we previously reported [40], the appearance of a red color was directly observed during the expression and the extraction steps of rFNIII9/10-tagged protein.…”

“…Sci. 2021, 22, x FOR PEER REVIEW 3 of 18 tag reported in our previous studies [40,41]. This system has demonstrated its performance in real time monitoring of rFNIII9/10 production and purification steps allowing high degree of purity and improved evaluating strategies.…”

“…The key advantage of this technology is that enabling an easy monitoring of production and purification process and our previous results showed a high purity purification using the double-purification system [40].…”

CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag

“…For that, researchers usually have to repeat several chromatography steps sequentially based on different principles, including affinity purification, ion exchange chromatography and size exclusion chromatography. In such a case, “dual tagging” or “multi tags” (e.g., FLAG tag and His 10 tag fused at the N- and C-terminus of the target protein, respectively) are often designed to help improve purity and reduce purification steps [2,9–12]. Considering the compatibility between tags and target proteins, new affinity purification systems with high efficiency and versatility are still highly needed.…”